Oxide Layers on Silicone Substrates for Effective Confocal Laser Microscopy

ABSTRACT

Methods of performing confocal laser microscopy on a polymer array disposed on a silicon wafer substrate, the method comprising the steps of providing a silicon wafer substrate having a top side and a bottom side, coating the top side of the silicon wafer with an oxide coating to provide an oxide coated wafer, covalently coupling a plurality of probes to the top side of the coated wafer to provide a fixed polymer array, hybridizing the fixed polymer array with a plurality of labeled ligands, and assaying for one or more hybridized ligands using confocal laser fluorescence microscopy to detect hybridization are provided.

FIELD OF THE INVENTION

The present invention relates to coated silicon substrates useful forarray synthesis and subsequent fluorescence analysis of arrays.

BACKGROUND OF THE INVENTION

Methods for synthesizing a variety of different types of polymers arewell known in the art. For example, the “Merrifield” method, describedin Atherton et a., “Solid Phase Peptide Synthesis,” IRL Press, 1989, hasbeen used to synthesize peptides on a solid support. In the Merrifieldmethod, an amino acid is covalently bonded to a support made of aninsoluble polymer or other material. Another amino acid with an alphaprotecting group is reacted with the covalently bonded amino acid toform a dipeptide. After washing, the protecting group is removed and athird amino acid with an alpha protecting group is added to thedipeptide. This process is continued until a peptide of a desired lengthand sequence is obtained.

Methods have also been developed for producing large arrays of polymersequences on solid substrates. These large “array” of polymer sequenceshave wide ranging applications and are of substantial importance to thepharmaceutical, biotechnology and medical industries. For example, thearrays may be used in screening large numbers of molecules forbiological activity, i.e., receptor binding capability. Alternatively,arrays of oligonucleotide probes can be used to identify mutations inknown sequences, as well as in methods for de novo sequencing of targetnucleic acids.

SUMMARY OF THE INVENTION

Embodiments of the present invention are based in part on the discoverythat a variety of silicon substrates comprising an oxide layer aresuitable for array synthesis and subsequent fluorescence analysis. Avariety of silicon substrates were investigated and determined to besuitable to support silanation and non-photochemical methods ofphosphoramidite-based probe synthesis, generating results that werecomparable to results obtained using fused silica.

The present invention provides methods of performing confocal lasermicroscopy on a polymer array disposed on a silicon wafer substrate. Incertain embodiments, a method of the invention includes providing asilicon wafer substrate having a top side and a bottom side, coating thetop side of the silicon wafer with an oxide coating to provide an oxidecoated wafer, covalently coupling a plurality of probes to the top sideof the coated wafer to provide a fixed polymer array, hybridizing thefixed polymer array with a plurality of labeled ligands, and assayingfor one or more hybridized ligands using confocal laser fluorescencemicroscopy to detect hybridization. Certain aspects of the inventioninclude applying BisB to the oxide coating.

In other embodiments, the present invention provides a method ofperforming confocal laser microscopy on a polymer array disposed on asilicon wafer substrate including the steps of providing a silicon wafersubstrate having a top side and a bottom side, coating the top side ofthe substrate with a transparent oxide layer to provide an oxide coatedwafer, depositing a reactive functional group comprising a labileprotecting group substantially uniformly across the transparent oxidelayer, selectively removing one or more of the labile protecting groupsfrom predefined regions of the wafer to provide exposed functionalgroups in said predefined regions, reacting the exposed functionalgroups with a monomer comprising a reactive functional group and alabile protecting group, repeating the steps of selectively removing andreacting to produce said polymer array, hybridizing the polymer arraywith a plurality of ligands, and assaying for one or more hybridizedligands using a confocal laser fluorescence microscopy to detecthybridization.

Certain aspects of the invention provide an oxide layer having athickness of at least 3,500 angstroms or having a thickness of at least35,000 angstroms. Other aspects of the invention provide that a labileprotecting group is an acid labile protecting group such as adimethoxytrityl group. Other aspects of the invention provide that anacid labile protecting group is removed by activating a photoacidgenerator with light of an appropriate wavelength to produce acid. Aphotoacid generator includes an ionic photoacid generator such as anonium salt such as bis-(4-t-butyl phenyl) iodonium PF₆ ⁻, or a non-ionicphotoacid generator such as 2,6-dinitrobenzyl tosylate.

Certain aspects of the invention provide that a labile protective groupis a photolabile protecting group. Labile protecting groups includeMeNPOC. In accordance with an aspect of the present invention, moreefficient photolabile protecting groups can also be used. It has beendiscovered in accordance with the present invention that to achievesuitable primer purity and quantity, a highly-efficient photogroup (>90%average stepwise coupling efficiency) is preferred, such as NPPOC orMBPMOC:

Both NNPOC and MBPMOC give greater than 90% stepwise coupling. Otheraspects of the invention provide that a monomer is a nucleotide, anucleic acid, an amino acid or a peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 depicts the site density of silicon wafers coated with BisB. Thedensity of the silicon wafers was 2.33 g/cm³, and the thickness of thesilicon wafers was approximately 400 to 700 μm.

FIG. 2 depicts TCA 6-mers on fused silica and oxide silicone.

FIG. 3 depicts dosage response curves of a photoacid generator (PAG) onoxide silicon BisB.

FIG. 4 depicts T-6-mers (PAG) with no base on oxide silicon and fusedsilica.

FIG. 5 depicts 100 mm Si wafer testing (BisB) using Cy3 stain galvoscans.

FIG. 6 depicts 100 mm Si wafer testing (BisB) using Cy3 stain Axonscans.

FIG. 7 depicts 100 mm Si wafer testing (BisB) using Cy3 stain galvoscans showing oxide thickness.

FIG. 8 depicts a 2 nM dual label target (BisB oxide-Si substrate)fluorescein channel, front side scan.

FIG. 9 depicts 100 mm Si wafer testing (BisB) using MeNPOC hexamers.MeNPOC stepwise deprotection was reduced to 55% on native oxide. Othersurfaces compared well to a fused-silica (FS) control, and, withoutintending to be bound by theory, each surface tested should produce goodsingle-MeNPOC experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used herein, the singular forms “a,” “an,” and “the” include, but arenot limited to, plural references unless the context clearly dictatesotherwise. For example, the term “an agent” includes, but is not limitedto, a plurality of agents, including mixtures thereof.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5 and 6. This appliesregardless of the breadth of the range.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry and immunology, which are withinthe skill of the art. Such conventional techniques include polymer arraysynthesis, hybridization, ligation, and detection of hybridization usinga label. Specific illustrations of suitable techniques can be had byreference to the description provided below. However, other equivalentconventional procedures can, of course, also be used. Such conventionaltechniques and descriptions can be found in standard laboratory manualssuch as Genome Analysis: A Laboratory Manual Series (Vols. I-IV), UsingAntibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer:A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (allfrom Cold Spring Harbor Laboratory Press), Stryer, L. (1995)Biochemistry (4th Ed.) Freeman, New York, Gait, “OligonucleotideSynthesis: A Practical Approach” 1984, IRL Press, London, Nelson and Cox(2000), Lehninger, Principles of Biochemistry 3rd Ed., W. H. FreemanPub., New York, N.Y. and Berg et al. (2002) Biochemistry, 5th Ed., W. H.Freeman Pub., New York, N.Y., all of which are herein incorporated intheir entirety by reference for all purposes.

The present invention can employ solid substrates, including arrays incertain embodiments. Methods and techniques applicable to polymer arraysynthesis have been described in U.S. Ser. No. 09/536,841, WO 00/58516,U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261,5,405,783, 5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681,5,550,215, 5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711,5,631,734, 5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659,5,936,324, 5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601,6,033,860, 6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, inPCT Applications Nos. PCT/US99/00730 (International Publication No. WO99/36760) and PCT/US01/04285 (International Publication No. WO01/58593), each of which is incorporated herein by reference in itsentirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165 and 5,959,098, each of which is incorporated herein byreference in its entirety for all purposes. Nucleic acid arrays aredescribed in many of the above patents, but the same techniques areapplied to polypeptide arrays.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring, and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822, which are all incorporated by reference in their entiretyfor all purposes. Genotyping and uses therefore are shown in U.S. Ser.Nos. 60/319,253, 10/013,598 (U.S. Patent Application Publication20030036069), and U.S. Pat. Nos. 5,856,092, 6,300,063, 5,858,659,6,284,460, 6,361,947, 6,368,799 and 6,333,179, which are incorporated byreference in their entirety for all purposes. Other uses are embodied inU.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and6,197,506, which are incorporated by reference in their entirety for allpurposes.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, each of which is incorporated herein by reference in itsentirety for all purposes. The sample may be amplified on the array.See, for example, U.S. Pat. No. 6,300,070 and U.S. Ser. No. 09/513,300,which are incorporated herein by reference in their entirety for allpurposes.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace (1989) Genomics 4:560, Landegren et al.(1988) Science 241:1077 and Barringer et al. (1990) Gene 89:117),transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci.USA 86:1173 and WO88/10315), self-sustained sequence replication(Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA, 87:1874 andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplication (NABSA). Each of the abovereferences is incorporated herein by reference in its entirety for allpurposes. (See, U.S. Pat. Nos. 5,409,818, 5,554,517, and 6,063,603, eachof which is incorporated herein by reference in its entirety for allpurposes). Other amplification methods that may be used are describedin, U.S. Pat. Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No.09/854,317. Each of the above references is incorporated herein byreference in its entirety.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al. (2001)Genome Research 11:1418, in U.S. Pat. Nos. 6,361,947, 6,391,592 and U.S.Ser. Nos. 09/916,135, 09/920,491 (U.S. Patent Application Publication20030096235), Ser. No. 09/910,292 (U.S. Patent Application Publication20030082543), and Ser. No. 10/013,598, each of which is incorporatedherein by reference in its entirety.

Numerous methods for conducting polynucleotide hybridization assays havebeen well developed. Hybridization assay procedures and conditions willvary depending on the application and are selected in accordance withthe general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (Academic Press, Inc., SanDiego, Calif., 1987); Young and Davism, Proc. Natl. Acad. Sci. USA,80:1194 (1983). Methods and apparatus for carrying out repeated andcontrolled hybridization reactions have been described in U.S. Pat. Nos.5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of whichis hereby incorporated by reference in its entirety.

The present invention contemplates detection of hybridization between aligand and its corresponding receptor by generation of specific signals.See U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758;5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639;6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and in PCTApplication PCT/US99/06097 (published as WO99/47964), each of which alsois hereby incorporated by reference in its entirety. Each of thesereferences is incorporated herein by reference in its entirety.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Ser. No. 60/364,731 and inPCT Application PCT/US99/06097 (published as WO99/47964), each of whichalso is hereby incorporated by reference in its entirety.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001). See U.S.Pat. No. 6,420,108. Each of these references is incorporated herein byreference in its entirety.

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170. Each ofreferences is incorporated herein by reference in its entirety.

Light patterns can also be generated using Digital Micromirrors, LightCrystal on Silicon (LCOS), light valve arrays, laser beam patterns andother devices suitable for direct-write photolithography. See. e.g.,U.S. Pat. Nos. 6,271,957 and 6,480,324, incorporated herein by referencein their entirety for all purposes.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing biological information over networks suchas the internet as shown in U.S. Ser. Nos. 10/197,621, 10/063,559(United States Publication No. 20020183936), Ser. Nos. 10/065,856,10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

The following definitions are used, unless otherwise described.

An “array,” as defined herein, includes but is not limited to apreselected collection of different polymer sequences or probes whichare associated with a surface of a substrate. An array may includepolymers of a given length having all possible monomer sequences made upof a specific basis set of monomers, or a specific subset of such anarray. For example, an array of all possible oligonucleotides of length8 includes 65,536 different sequences. However, as noted above, anoligonucleotide array also may include only a subset of the complete setof probes. Similarly, a given array may exist on more than one separatesubstrate, e.g., where the number of sequences necessitates a largersurface area in order to include all of the desired polymer sequences.

A “functional group,” as used herein, includes but is not limited to areactive chemical moiety present on a given monomer, polymer orsubstrate surface. Examples of functional groups include, e.g., the 3′and 5′ hydroxyl groups of nucleotides and nucleosides, as well as thereactive groups on the nucleobases of the nucleic acid monomers, e.g.,the exocyclic amine group of guanosine, as well as amino and carboxylgroups on amino acid monomers.

A “monomer” or “building block,” as used herein, includes but is notlimited to a member of the set of smaller molecules which can be joinedtogether to form a larger molecule or polymer. The set of monomersincludes but is not restricted to, for example, the set of commonL-amino acids, the set of D-amino acids, the set of natural or syntheticamino acids, the set of nucleotides (both ribonucleotides anddeoxyribonucleotides, natural and unnatural) and the set of pentoses andhexoses. As used herein, monomer refers to any member of a basis set forsynthesis of a larger molecule. A selected set of monomers forms a basisset of monomers. For example, the basis set of nucleotides includes A, T(or U), G and C. In another example, dimers of the 20 naturallyoccurring L-amino acids form a basis set of 400 monomers for synthesisof polypeptides. Different basis sets of monomers may be used in any ofthe successive steps in the synthesis of a polymer. Furthermore, each ofthe sets may include protected members which are modified aftersynthesis.

A “feature,” as used herein, includes but is not limited to a selectedregion on a surface of a substrate in which a given polymer sequence iscontained. Thus, where an array contains, e.g., 100,000 differentpositionally distinct polymer sequences on a single substrate, therewill be 100,000 features.

An “edge,” as used herein, includes but is not limited to a boundarybetween two features on a surface of a substrate. The sharpness of thisedge, in terms of reduced bleed-over from one feature to another, istermed the “contrast” between the two features.

A “protecting group,” as used herein, includes but is not limited to amaterial which is chemically bound to a reactive functional group on amonomer unit or polymer and which protective group may be removed uponselective exposure to an activator such as a chemical activator, oranother activator, such as electromagnetic radiation or light,especially ultraviolet and visible light. Protecting groups that areremovable upon exposure to electromagnetic radiation, and in particularlight, are termed “photolabile protecting groups.”

Halo is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, aralkyl,alkylaryl, and the like denote both straight and branched alkyl groups,but reference to an individual radical such as “propyl” embraces onlythe straight chain radical, a branched chain isomer such as “isopropyl”being specifically referred to. Aryl includes a phenyl radical or anortho-fused, bicyclic, carbocyclic radical having about nine to ten ringatoms in which at least one ring is aromatic. Heteroaryl encompasses aradical attached via a ring carbon of a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is H, O, (C,—C₄)alkyl,phenyl or benzyl, as well as a radical of an ortho-fused, bicyclicheterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene or tetramethylene diradical thereto.

An “alkyl,” as used herein, refers without limitation to a straightchain, branched or cyclic chemical groups containing only carbon andhydrogen. Alkyl groups include, without limitation, ethyl, propyl,butyl, pentyl, cyclopentyl and 2-methylbutyl. Alkyl groups areunsubstituted or substituted with one or more substituents (e.g.,halogen, alkoxy, amino).

An “alkylene,” as used herein, refers without limitation to a straightchain, branched or cyclic chemical group containing only carbon andhydrogen. Alkyl groups include, without limitation, ethylene, propylene,butylene, pentylene, and 2-methylbutylene. Alkyl groups areunsubstituted or substituted with one or more substituents (e.g.,halogen, alkoxy, amino).

An “aryl,” as used herein, refers without limitation to a monovalent,unsaturated, aromatic carbocyclic group. Aryl groups include, withoutlimitation, phenyl, naphthyl, anthryl and biphenyl. Aryl groups areunsubstituted or substituted with 1 or more substituents (e.g. halogen,alkoxy, amino). “Arylene” refers to a divalent aryl group.

An “amido,” as used herein, refers without limitation to a chemicalgroup having the structure —C(O)NR₃—, wherein R₃ is hydrogen, alkyl oraryl. Preferably, the amido group is of the structure —C(O)NR₃— where R₃is hydrogen or alkyl having from about 1 to about 6 carbon atoms. Morepreferably, the amido alkyl group is of the structure —C(O)NH—.

An “alkanoyl,” as used herein, refers without limitation to a chemicalgroup having the structure —(CH₂)_(n)C(O)—, wherein n is an integerranging from 0 to about 10. Preferably, the alkanoyl group is of thestructure —(CH₂)_(n)C(O)—, wherein n is an integer ranging from about 2to about 10. More preferably, the alkanoyl group is of the structure—(CH₂)_(n)(O)—, wherein n is an integer ranging from about 2 to about 6.Most preferably, the alkanoyl group is of the structure —CH₂C(O)—.

An “alkyl amido,” as used herein, refers without limitation to achemical group having the structure —R₄C(O)NR₃—, wherein R₃ is hydrogen,alkyl or aryl, and R₄ is alkylene or arylene. Preferably, the alkylamido group is of the structure —(CH₂)_(n)C(O)NH—, wherein n is aninteger ranging from about 1 to about 10. More preferably, n is aninteger ranging from about 1 to about 6. Most preferably, the alkylamido group has the structure —(CH₂)₂C(O)NH— or the structure—CH₂C(O)NH—.

An “N-amido alkyl,” as used herein, refers without limitation to achemical group having the structure —C(O)NR₃R₄—, wherein R₃ is hydrogen,alkyl or aryl, and R₄ is alkylene or arylene. Preferably, the N-amidoalkyl group is of the structure —C(O)NH(CH₂)_(n)R₅—, wherein n is aninteger ranging from about 2 to about 10, and R₅ is O, NR₆, or C(O), andwherein R₆ is hydrogen, alkyl or aryl. More preferably, the N-amidoalkyl group is of the structure —C(O)NH(CH₂)_(n)N(H)—, wherein n is aninteger ranging from about 2 to about 6. Most preferably, the N-amidoalkyl group is of the structure —C(O)NH(CH₂)₄N(H)—.

An “alkynyl alkyl,” as used herein, refers without limitation to achemical group having the structure —CC—R₄—, wherein R₄ is alkyl oraryl. Preferably, the alkynyl alkyl group is of the structure—CC—(CH₂)_(n)R₅—, wherein n is an integer ranging from 1 to about 10,and R₅ is O, NR₆ or C(O), wherein R₆ is hydrogen, alkyl or aryl. Morepreferably, the alkynyl alkyl group is of the structure—CC—(CH₂)_(n)N(H)—, wherein n is an integer ranging from 1 to about 4.Most preferably, the alkynyl alkyl group is of the structure—CC—CH₂N(H)—.

An “alkenyl alkyl,” as used herein, refers without limitation to achemical group having the structure —CH═CH—R₄—, wherein R₄ is a bond,alkyl or aryl. Preferably, the alkenyl alkyl group is of the structure—CH═CH—(CH₂)nR₅—, wherein n is an integer ranging from 0 to about 10,and R₅ is O, NR₆, C(O) or C(O)NR₆, wherein R₆ is hydrogen, alkyl oraryl. More preferably, the alkenyl alkyl group is of the structure—CH═CH— (CH₂)_(n)C(O)NR₆—, wherein n is an integer ranging from 0 toabout 4. Most preferably, the alkenyl alkyl group is of the structure—CH═CH—C(O)N(H)—.

A “functionalized alkyl,” as used herein, refers without limitation to achemical group of the structure —(CH₂)_(n)R₇—, wherein n is an integerranging from 1 to about 10, and R₇ is O, S, NH or C(O). Preferably, thefunctionalized alkyl group is of the structure —(CH₂)_(n)C(O)—, whereinn is an integer ranging from 1 to about 4. More preferably, thefunctionalized alkyl group is of the structure —CH₂C(O)—.

An “alkoxy,” as used herein, refers without limitation to a chemicalgroup of the structure —O(CH₂)_(n)R₈—, wherein n is an integer rangingfrom 2 to about 10, and R₈ is a bond, O, S, NH or C(O). Preferably, thealkoxy group is of the structure —O(CH₂)n, wherein n is an integerranging from 2 to about 4. More preferably, the alkoxy group is of thestructure —OCH₂CH₂—.

An “alkyl thio,” as used herein, refers without limitation to a chemicalgroup of the structure —S(CH₂)_(n)R₈—, wherein n is an integer rangingfrom 1 to about 10, and R₈ is a bond, O, S, NH or C(O). Preferably, thealkyl thio group is of the structure —S(CH₂)_(n), wherein n is aninteger ranging from 2 to about 4. More preferably, the thio group is ofthe structure —SCH₂CH₂C(O)—.

An “amino alkyl,” as used herein, refers without limitation to achemical group having an amino group attached to an alkyl group.Preferably an amino alkyl is of the structure —(CH)_(n)NH—, wherein n isan integer ranging from about 2 to about 10. More preferably it is ofthe structure —(CH₂)_(n)NH—, wherein n is an integer ranging from about2 to about 4. Most preferably, the amino alkyl group is of the structure—(CH₂)₂NH—.

The term “nucleic acid,” as used herein, includes, but is not limitedto, a polymer comprising two or more nucleotides and includes single-,double- and triple stranded polymers. As used herein, the term“nucleotide” refers without limitation to both naturally occurring andnon-naturally occurring compounds and comprises a heterocyclic base, asugar, and a linking group, preferably a phosphate ester. As usedherein, the term “nucleoside” refers to both naturally occurring andnon-naturally occurring compounds and comprises a heterocyclic base anda sugar.

Structural groups may be added to the ribosyl or deoxyribosyl unit ofthe nucleotide, such as a methyl or allyl group at the 2′-O position ora fluoro group that substitutes for the 2′-O group. The linking group,such as a phosphodiester, of the nucleic acid may be substituted ormodified, for example with methyl phosphonates or O-methyl phosphates.Bases and sugars can also be modified, as is known in the art. “Nucleicacid,” for the purposes of this disclosure, also includes “peptidenucleic acids” in which native or modified nucleic acid bases areattached to a polyamide backbone.

The term “oligonucleotide,” sometimes referred to as “polynucleotide,”includes, but is not limited to, a nucleic acid ranging from at least 5,10, or 20 bases long and may be up to 20, 50, 100, 1,000, or 5,000 baseslong and/or a compound that specifically hybridizes to a polynucleotide.Oligonucleotides of the present invention include sequences ofdeoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and mimeticsthereof which may be isolated from natural sources, recombinantlyproduced or artificially synthesized. A further example of apolynucleotide of the present invention is a peptide nucleic acid (PNA).See U.S. Pat. No. 6,156,501, incorporated herein by reference in itsentirety for all purposes. The invention also encompasses situations inwhich there is a nontraditional base pairing such as Hoogsteen basepairing which has been identified in certain tRNA molecules andpostulated to exist in a triple helix. “Polynucleotide” and“oligonucleotide” are used interchangeably in this application.

The phrase “coupled to a support” includes, but is not limited to, beingbound directly or indirectly thereto including attachment by covalentbinding, hydrogen bonding, ionic interaction, hydrophobic interaction,or otherwise.

A “probe,” as defined herein, includes but is not limited to asurface-immobilized molecule that is recognized by a particular target.These may also be referred to as ligands. Examples of probes encompassedby the scope of this invention include, but are not limited to, agonistsand antagonists of cell surface receptors, toxins and venoms, viralepitopes, hormone receptors, peptides, peptidomimetics, enzymes, enzymesubstrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleicacids, oligosaccharides, proteins or monoclonal antibodies, natural ormodified, e.g., reshaped, chimeric, etc.

The terms “solid support,” “support,” and “substrate” as used herein areused interchangeably and include, but are not limited to, a material orgroup of materials having a rigid or semi-rigid surface or surfaces. Inmany embodiments, at least one surface of the solid support will besubstantially flat or planar, although in some embodiments it may bedesirable to physically separate synthesis regions for differentcompounds with, for example, wells, raised regions, pins, etchedtrenches, or the like. According to other embodiments, the solidsupport(s) will take the form of beads, resins gels, microspheres, orother geometric configurations. Preferred substrates generally compriseplanar crystalline substrates used in, e.g., the semiconductor andmicroprocessor industries, such as silicon, gallium arsenide and thelike, or crystalline substrates such as silica based substrates (e.g.glass, quartz, or the like). These substrates are generally resistant tothe variety of synthesis and analysis conditions to which they may besubjected. See U.S. Pat. No. 5,744,305 and U.S. Patent Appln. Pub.20040105932, each of which is incorporated herein by reference in itsentirety for all purposes, for exemplary substrates.

Individual planar substrates generally exist as wafers which can havevaried dimensions. As used herein, the term “wafer” generally referswithout limitation to a substantially flat sample of substrate fromwhich a plurality of individual arrays or chips may be fabricated. Theterms “array” or “chip” are used without limitation to refer to thefinal product of the individual array of polymer sequences, having aplurality of different positionally distinct polymer sequences coupledto the surface of the substrate. The size of a substrate wafer isgenerally defined by the number and nature of arrays that will beproduced from the wafer. For example, more complex arrays, e.g., arrayshaving all possible polymer sequences produced from a basis set ofmonomers and having a given length, will generally utilize larger areasand thus employ larger substrates, whereas simpler arrays may employsmaller surface areas, and thus, less substrate.

In certain aspects of the invention, silicon wafers can be used tofabricate high-density arrays of oligonucleotides using the techniquesdescribed herein. Certain advanced lithography equipment (e.g., astepper) is designed around the industry-standard round silicon wafersubstrate. Commercially available substrates are typically 5, 6 and 8inches in diameter. In contrast to fused silica, silicon wafers arethinner and non-transparent. The opacity of silicon requires thatphotochemistry and also scanning be performed “front side.” A number ofbulk physical properties, such as crystal lattice orientation, dopant,resistivity and insulating oxide layer thickness have been discovered inaccordance with the instant invention to not be critical to silanationor array synthesis. It was also discovered in accordance with thepresent invention that a variety of silicon substrates supportsilanation and non-photochemical methods of phosphoramidite-based probesynthesis with results comparable to fused silica. However, inaccordance with the present invention, it has been discovered thateffective confocal laser scanning has been found to be surprisinglydependent upon a suitable coating such as a layer of transparent oxide.

Typically, the substrate wafer will range in size of from about 1″×1″ toabout 12″×12″, and will have a thickness of from about 0.5 mm to about 5mm. Individual substrate segments which include the individual arrays,or in some cases a desired collection of arrays, are typically muchsmaller than the wafers, measuring from about 0.2 cm.×0.2 cm to about 5cm×5 cm. In particularly preferred aspects, the substrate wafer is about5″×5″ whereas the substrate segment is approximately 1.28 cm×1.28 cm.Although a wafer can be used to fabricate a single large substratesegment, typically, a large number of substrate segments will beprepared from a single wafer. For example, a wafer that is 5″×5″ can beused to fabricate upwards of 49 separate 1.28 cm×1.28 cm substratesegments. The number of segments prepared from a single wafer willgenerally vary depending upon the complexity of the array, and thedesired feature size.

Although primarily described in terms of flat or planar substrates, thepresent invention may also be practiced with substrates havingsubstantially different conformations. For example, the substrate mayexist as particles, strands, precipitates, gels, sheets, tubing,spheres, containers, capillaries, pads, slices, films, plates, slides,and the like. In a preferred alternate embodiment, the substrate is aglass tube or microcapillary. The capillary substrate providesadvantages of higher surface area to volume ratios, reducing the amountof reagents necessary for synthesis. Similarly, the higher surface tovolume ratio of these capillary substrates imparts more efficientthermal transfer properties. Additionally, preparation of the polymerarrays may be simplified through the use of these capillary basedsubstrates. For example, minimizing differences between the regions onthe array, or “cells,” and their “neighboring cells” is simplified inthat there are only two neighboring cells for any given cell (seediscussion below for edge minimization in chip design). Spatialseparation of two neighboring cells on an array merely involves theincorporation of a single blank cell, as opposed to full blank lanes asgenerally used in a flat substrate conformation. This substantiallyconserves the surface area available for polymer synthesis.Manufacturing design may also be simplified by the linear nature of thesubstrate. In particular, the linear substrate may be moved down asingle mask in a direction perpendicular to the length of the capillary.As it is moved, the capillary will encounter linear reticles(translucent regions of the mask), one at a time, thereby exposingselected regions within the capillary or capillary. This can allowbundling of parallel capillaries during synthesis wherein thecapillaries are exposed to thicker linear reticles, simultaneously, fora batch processing mode, or individual capillaries may be placed on amask having all of the linear reticles lined up so that the capillarycan be stepped down the mask in one direction. Subsequent capillariesmay be stepped down the mask at least one step behind the previouscapillary. This employs an assembly line structure to the substratepreparation process.

As an example, a standard optimization chip for detecting 36simultaneous mutations using a flat geometry chip and an optimizationtiling strategy, is 44×45 features (1980 probes and blanks), with 36blocks of 40 probes each (1440 probes), plus 15 blanks per block (540blank probes). A capillary format, however, can incorporate the samenumber of test probes in a smaller space. Specifically, in a capillarysubstrate, 36 strings of 40 probes will have only one blank spaceseparating each probe group (35 blank probes), for a total of 1475features.

Finally, linear capillary based substrates can provide the advantage ofreduced volume over flat geometries. In particular, typical capillarysubstrates have a volume in the 1-10 μl range, whereas typical flowcells for synthesizing or screening flat geometry chips have volumes inthe range of 100 μl.

A. Stripping and Rinsing

In one aspect of the present invention, oxide-coated wafers aresilanated as supplied from the wafer vendor without prior stripping. Inother aspects, in order to ensure maximum efficiency and accuracy insynthesizing polymer arrays, it is desirable to provide a cleansubstrate surface upon which the various reactions are to take place.Accordingly, in some processing embodiments of the present invention,the substrate is stripped to remove any residual dirt, oils or otherfluorescent materials which may interfere with the synthesis reactions,or subsequent analytical use of the array.

The process of stripping the substrate typically involves applying,immersing or otherwise contacting the substrate with a strippingsolution. Stripping solutions may be selected from a number ofcommercially available, or readily prepared chemical solutions used forthe removal of dirt and oils, which solutions are well known in the art.Particularly preferred stripping solutions are composed of a mixture ofconcentrated H₂SO₄ and H₂O₂. Such solutions are generally available fromcommercial sources, e.g., NANOSTRIPT™ from Cyantek Corp. (Fremont,Calif.). After stripping, the substrate is rinsed with water and inpreferred aspects, is then contacted with a solution of NaOH, whichresults in regeneration of an even layer of hydroxyl functional groupson the surface of the substrate. In this case, the substrate is againrinsed with water, followed by a rinse with HCl to neutralize anyremaining base, followed again by a water rinse. The various strippingand rinsing steps may generally be carried out using a spin-rinse-dryingapparatus of the type generally used in the semiconductor manufacturingindustry.

Gas phase cleaning and preparation methods may also be applied to thesubstrate wafers using, e.g., H₂O or O₂ plasma or reactive ion etching(RIE) techniques that are well known in the art.

B. Coating Layer

Embodiments of the present invention are based on the unexpected findingthat there is a strong correlation between array signal detection andoxide layer thickness. In accordance with a preferred embodiment,silicon wafers coated with a coating layer, e.g., an oxide layer, areused as substrates for array synthesis and subsequent fluorescenceanalysis. An oxide layer can be composed of conventional oxide materialssuch as silicon oxide (SiO), silicon dioxide (SiO₂), borophosphosilicateglass (BPSG), borosilicate glass (BSG), fluorosilicate glass (FSG),tetraethoxysilane (TEOS), and the like. Oxide layers are described inU.S. Pat. No. 6,191,046, incorporated herein by reference in itsentirety for all purposes.

In a certain embodiments, the coating layer, e.g., an oxide layer, has athickness of approximately at least 3,500 angstroms, 4,000 angstroms,4,500 angstroms, 5,000 angstroms, 5,500 angstroms, 6,000 angstroms,6,500 angstroms, 7,000 angstroms, 7,500 angstroms, 8,000 angstroms,8,500 angstroms, 9,000 angstroms, 9,500 angstroms, 10,000 angstroms,11,000 angstroms, 12,000 angstroms, 13,000 angstroms, 14,000 angstroms,15,000 angstroms, 16,000 angstroms, 17,000 angstroms, 18,000 angstroms,19,000 angstroms, 20,000 angstroms, 21,000 angstroms, 22,000 angstroms,23,000 angstroms, 24,000 angstroms, 25,000 angstroms, 26,000 angstroms,27,000 angstroms, 28,000 angstroms. 29,000 angstroms, 30,000 angstroms,40,000 angstroms, 45,000 angstroms, 50,000 angstroms, 55,000 angstroms,60,000 angstroms, 65,000 angstroms, 70,000 angstroms, 75,000 angstroms,80,000 angstroms, 85,000 angstroms, 90,000 angstroms, 95,000 angstroms,100,000 angstroms, or more. In a preferred embodiment, a coating layer(e.g., an oxide layer) is approximately at least 3,500 angstroms thick.In a particularly preferred embodiment, a coating layer (e.g., an oxidelayer) is approximately at least 35,000 angstroms thick.

In accordance with preferred aspects of the present invention, it iscontemplated that antireflective or adsorptive coatings can substitutefor an oxide coating to attain a robust fluorescence signal.Antireflective and/or adsorptive coatings are known in the art anddescribed in U.S. Pat. No. 6,156,149, incorporated herein by referencein its entirety for all purposes.

In certain aspects of the invention, a coating comprising a siliconcompound can be added to the substrates described herein. Such coatingscan be added to the substrate itself or to another coating layer.Suitable silicon compounds are described in U.S. Patent Appl. Pub. No.20010027187, incorporated herein by reference in its entirety for allpurposes.

The use of a fluorophore in front of a reflecting surface and theresulting interaction of standing waves as a function of the distance tothe reflector and the wavelength(s) of light is described in Lambacherand Fromherz (1996) Appl. Phys. A 63:207; Braun and Fromherz (1997)Appl. Phys. A 65:341; Braun and Fromherz (1998) Phys. Rev. Lett.81:5241; and Drexhage (1974) Prog. In Optics XII:163, each of which isincorporated herein by reference in its entirety for all purposes.

C. Derivatization

Following the optional step of cleaning and stripping of the substratesurface and the addition of a coating layer, the surface is derivatizedto provide sites or functional groups on the substrate surface forsynthesizing the various polymer sequences on that surface. Inparticular, derivatization provides reactive functional groups, e.g.,hydroxyl, carboxyl, amino groups or the like, to which the firstmonomers in the polymer sequence may be attached. In preferred aspects,the substrate surface is derivatized using silane in either water orethanol. Preferred silanes include mono- and dihydroxyalkylsilanes,which provide a hydroxyl functional group on the surface of thesubstrate. Also preferred are aminoalkyltrialkoxysilanes which can beused to provide the initial surface modification with a reactive aminefunctional group. Particularly preferred are3-aminopropyltriethoxysilane and 3-aminopropyltrimethoxysilane (“APS”).Derivatization of the substrate using these latter amino silanesprovides a linkage that is stable under synthesis conditions and finaldeprotection conditions (for oligonucleotide synthesis, this linkage istypically a phosphoramidate linkage, as compared to the phosphodiesterlinkage where hydroxyalkylsilanes are used). Additionally, this aminosilane derivatization provides several advantages over derivatizationwith hydroxyalkylsilanes. For example, the aminoalkyltrialkoxysilanesare inexpensive and can be obtained commercially in high purity from avariety of sources, the resulting primary and secondary amine functionalgroups are more reactive nucleophile than hydroxyl groups, theaminoalkyltrialkoxysilanes are less prone to polymerization duringstorage, and they are sufficiently volatile to allow application in agas phase in a controlled vapor deposition process.

Additionally, silanes can be prepared having protected or “masked”hydroxyl groups and which possess significant volatility. As such, thesesilanes can be readily purified by, e.g., distillation, and can bereadily employed in gas-phase deposition methods of silanating substratesurfaces. After coating these silanes onto the surface of the substrate,the hydroxyl groups may be deprotected with a brief chemical treatment,e.g., dilute acid or base, which will not attack the substrate-silanebond, so that the substrate can then be used for polymer synthesis.Examples of such silanes include acetoxyalkylsilanes, such asacetoxyethyltrichlorosilane, acetoxypropyltrimethoxysilane, which may bedeprotected after application using, e.g., vapor phase ammonia andmethylamine or liquid phase aqueous or ethanolic ammonia andalkylamines. Epoxyalkylsilanes may also be used, such asglycidoxypropyltrimethoxysilane which may be deprotected using, e.g.,vapor phase HCl, trifluoroacetic acid or the like, or liquid phasedilute HCl.

The physical operation of silanation of the substrate generally involvesdipping or otherwise immersing the substrate in the silane solution.Following immersion, the substrate is generally spun as described forthe substrate stripping process, i.e., laterally, to provide a uniformdistribution of the silane solution across the surface of the substrate.This ensures a more even distribution of reactive functional groups onthe surface of the substrate. Following application of the silane layer,the silanated substrate may be baked to polymerize the silanes on thesurface of the substrate and improve the reaction between the silanereagent and the substrate surface. Baking typically takes place attemperatures in the range of from 90° C. to 120° C., with 110° C. beingmost preferred, for a time period of from about 1 minute to about 10minutes, with 5 minutes being preferred.

In alternative aspects, as noted above, the silane solution may becontacted with the surface of the substrate using controlled vapordeposition methods or spray methods. These methods involve thevolatilization or atomization of the silane solution into a gas phase orspray, followed by deposition of the gas phase or spray upon the surfaceof the substrate, usually by ambient exposure of the surface of thesubstrate to the gas phase or spray. Vapor deposition typically resultsin a more even application of the derivatization than simply immersingthe substrate into the solution.

The efficacy of the derivatization process, e.g., the density anduniformity of functional groups on the substrate surface, may generallybe assessed by adding a fluorophore which binds the reactive groups,e.g., a fluorescent phosphoramidite such as FLUOREPRIME™ from PfizerInc. (New York, N.Y.), FLUOREDITE™ from Millipore Corp. (Billerica,Mass.) or FAM, and ascertaining the relative fluorescence across thesurface of the substrate.

D. Synthesis

General methods for the solid phase synthesis of a variety of polymertypes have been previously described. Methods of synthesizing arrays oflarge numbers of polymer sequences, including oligonucleotides andpeptides, on a single substrate have also been described. See U.S. Pat.Nos. 5,143,854 and 5,384,261 and Published PCT Application No WO92/10092, each of which is incorporated herein by reference in itsentirety for all purposes.

The synthesis of oligonucleotides on the surface of a substrate can becarried out using light directed methods as described in, e.g., U.S.Pat. Nos. 5,143,854 and 5,384,261 and Published PCT Application No WO92/10092, or mechanical synthesis methods as described in U.S. Pat. No.5,384,261 and Published PCT Application No. 93/09668, each of which isincorporated herein by reference in its entirety for all purposes. Inpreferred embodiments, photochemical steps, and in particular photoacidgenerator (PAG) synthesis techniques are preformed “front side” on asubstrate having a front side and a back side. In particular, theselight-directed or photolithographic synthesis methods involve aphotolysis step and a chemistry step. The substrate surface, prepared asdescribed herein, comprises functional groups on its surface. Thesefunctional groups are protected by photolabile protecting groups, i.e.,“photoprotected,” also as described herein. During the photolysis step,portions of the surface of the substrate are exposed to light or otheractivators to activate the functional groups within those portions,i.e., to remove photoprotecting groups. The substrate is then subjectedto a chemistry step in which chemical monomers that are photoprotectedat one or more functional groups are then contacted with the surface ofthe substrate. These monomers bind to the activated portion of thesubstrate through an unprotected functional group.

Subsequent activation and coupling steps couple monomers to otherpreselected regions, which may overlap with all or part of the firstregion. The activation and coupling sequence at each region on thesubstrate determines the sequence of the polymer synthesized thereon. Inparticular, light is shown through the photolithographic masks which aredesigned and selected to expose and thereby activate a first particularpreselected portion of the substrate. Monomers are then coupled to allor part of this portion of the substrate. The masks used and monomerscoupled in each step can be selected to produce arrays of polymershaving a range of desired sequences, each sequence being coupled to adistinct spatial location on the substrate which location also dictatesthe polymer's sequence. The photolysis steps and chemistry steps arerepeated until the desired sequences have been synthesized upon thesurface of the substrate.

Basic strategy for light directed synthesis of oligonucleotides on aVLSIPS™ Array is described in U.S. Patent Appl. Pub. No. 20040105932,incorporated herein by reference in its entirety for all purposes.Briefly, the surface of a substrate or solid support, modified withphotosensitive protecting groups is illuminated through aphotolithographic mask, yielding reactive hydroxyl groups in theilluminated regions. A selected nucleotide, typically in the form of a3′-O-phosphoramidite-activated deoxynucleoside (protected at the 5′hydroxyl with a photosensitive protecting group), is then presented tothe surface and coupling occurs at the sites that were exposed to light.Following capping and oxidation, the substrate is rinsed and the surfaceis illuminated through a second mask, to expose additional hydroxylgroups for coupling. A second selected nucleotide (e.g., 5′-protected,3′-O-phosphoramidite-activated deoxynucleoside) is presented to thesurface. The selective deprotection and coupling cycles are repeateduntil the desired set of products is obtained (Pease et al. (1994) Proc.Natl. Acad. Sci. USA 91:5022, incorporated herein by reference in itsentirety for all purposes. Since photolithography is used, the processcan be readily miniaturized to generate high density arrays ofoligonucleotide probes. Furthermore, the sequence of theoligonucleotides at each site is known. Such photolithographic methodsare also described in U.S. Pat. Nos. 5,143,854 and 5,489,678 andPublished PCT Application No. WO 94/10128 each of which is incorporatedherein by reference in its entirety for all purposes. In the large scaleprocesses of the present invention, it is typically preferred to utilizephotolithographic synthesis methods.

Using the above described methods, arrays may be prepared having allpolymer sequences of a given length which are composed of a basis set ofmonomers. Such an array of oligonucleotides, made up of the basis set offour nucleotides, for example, would contain up to 4^(n)oligonucleotides on its surface, where n is the desired length of theoligonucleotide probe. For an array of 8-mer or 10-mer oligonucleotides,such arrays could have upwards of about 65,536 and 1,048,576 differentoligonucleotides, respectively. Generally, where it is desired toproduce arrays having all possible polymers of length n, a simple binarymashing strategy can be used, as described in U.S. Pat. No. 5,143,854,incorporated herein by reference in its entirety for all purposes.

Alternate masking strategies can produce arrays of probes which containa subset of polymer sequences, i.e., polymers having a given subsequenceof monomers, but are systematically substituted at each position witheach member of the basis set of monomers. In the context ofoligonucleotide probes, these alternate synthesis strategies may be usedto lay down or “tile” a range of probes that are complementary to, andspan the length of a given known nucleic acid segment. The tilingstrategy will also include substitution of one or more individualpositions within the sequence of each of the probe groups with eachmember of the basis set of nucleotides. These positions are termed“interrogation positions.” By reading the hybridization pattern of thetarget nucleic acid, one can determine if and where any mutations lie inthe sequence, and also determine what the specific mutation is byidentifying which base is contained within the interrogation position.Tiling methods and strategies are discussed in substantial detail inU.S. patent application Ser. No. 08/143,312 filed Oct. 26, 1993, andincorporated herein by reference in its entirety for all purposes.

Tiled arrays may be used for a variety of applications, such asidentifying mutations within a known oligonucleotide sequence or“target.” Specifically, the probes on the array will have a subsequencewhich is complementary to a known nucleic acid sequence, but wherein atleast one position in that sequence has been systematically substitutedwith the other three nucleotides.

Use of photolabile protecting groups during polymer synthesis has beenpreviously reported, as described above. Preferred photolabileprotecting groups generally have the following characteristics: theyprevent selected reagents from modifying the group to which they areattached; they are stable to synthesis reaction conditions (that is,they remain attached to the molecule); they are removable underconditions that minimize potential adverse effects upon the structure towhich they are attached; and, once removed, they do not reactappreciably with the surface or surface bound oligomer. In someembodiments, liberated byproducts of the photolysis reaction can berendered non-reactive toward the growing oligomer by adding a reagentthat specifically reacts with the byproduct.

The removal rate of the photolabile protecting groups generally dependsupon the wavelength and intensity of the incident radiation, as well asthe physical and chemical properties of the protecting group itself.Preferred protecting groups are removed at a faster rate and with alower intensity of radiation. Generally, photoprotecting groups thatundergo photolysis at wavelengths in the range from 300 nm toapproximately 450 nm are preferred.

Generally, photolabile or photosensitive protecting groups includeortho-nitrobenzyl and ortho-nitrobenzyloxycarbonyl protecting groups.The use of these protecting groups has been proposed for use inphotolithography for electronic device fabrication (see, e.g.,Reichmanis et al. (1985) J. Polymer Sci. Polymer Chem. Ed. 23:1,incorporated herein by reference in its entirety for all purposes).

Examples of additional photosensitive protecting groups which may beused in the light directed synthesis methods herein described, include,e.g., 1-pyrenylmethyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl,4-methoxyphenacyloxycarbonyl, 3′-methoxybenzoinyloxycarbonyl,3′,5′-dimethoxybenzoinyloxycarbonyl2′,3′-dimethoxy-benzoinyloxycarbonyl,2′,3′-(methylenedioxy)benzoinyloxycarbonyl,N-(5-bromo-7-nitroindolinyl)carbonyl 3,5-dimethoxybenzyloxycarbony-1,α-(2-methylene-anthraquinone)oxycarbonyl and the like.

Particularly preferred photolabile protecting groups for protection ofeither the 3′ or 5′-hydroxyl-groups of nucleotides or nucleic acidpolymers include the o-nitrobenzyl protecting groups described inPublished PCT Application No. WO 92/10092, incorporated herein byreference in its entirety for all purposes. These photolabile protectinggroups include, e.g., (2-nitro-naphthalen-1-yl)-phenylmethylcarbonyl(NNPOC), 94′-methoxy-3-nitro-biphenyl-4-yl)-phenylmethylcarbonyl(MBPMOC), nitroveratryloxycarbonyl (NVOC), nitropiperonyl oxycarbonyl(NPOC), α-methyl-nitroveratryloxycarbonyl (MeNVOC),α-methyl-nitropiperonyloxycarbonyl (MeNPOC), 1-pyrenylmethyloxycarbonyl(PYMOC), and the benzylic forms of each of these (i.e., NNP, MBPM, NV,NP, MeNV, MeNP and PYM, respectively), with MeNPOC, NPPOC and MBPMOCbeing most preferred.

Protection strategies may be optimized for different phosphoramiditenucleosides to enhance synthesis efficiency. Examples of such optimizedsynthesis methods are reported in, e.g., U.S. patent application Ser.No. 08/445,332 filed May 19, 1995, incorporated herein by reference inits entirety for all purposes. Generally, these optimization methodsinvolve selection of particular protecting groups for protection of theO⁶ group of guanosine, which can markedly improve coupling efficienciesin the synthesis of guanosine containing oligonucleotides. Similarly,selection of the appropriate protecting group for protection of the N²group of guanosine can also result in such an improvement in the absenceof protection of the O⁶ group. For example, suitable protecting groupsfor protection of the N² group, where the O⁶ group is also protected,include, e.g., mono- or diacyl protecting groups, triarylmethylprotecting groups, e.g., DMT and MMT, and amidine type protectinggroups, e.g., N,N-dialkylformamidines. Particularly preferred protectinggroups for the N² group include, e.g., DMT, DMF, PAC, Bz and Ibu.

Protection of the O⁶ group will generally be carried out using carbamateprotecting groups such as —C(O)NX₂, where X is alkyl, or aryl; or theprotecting group —CH₂CH₂Y, where Y is an electron withdrawing group suchas cyano, p-nitrophenyl, or alkyl- or aryl-sulfonyl; and aryl protectinggroups. In a particularly preferred embodiment, the O⁶ group isprotected using a diphenylcarbamoyl protecting group (DPC).

Alternatively, improved coupling efficiencies may be achieved byselection of an appropriate protecting group for only the N² group. Forexample, where the N²-PAC protecting group is substituted with an Ibuprotecting group, a substantial improvement in coupling efficiency isseen, even without protection of the O⁶ group.

A variety of modifications can be made to the above-described synthesismethods. For example, in some embodiments, it may be desirable todirectly transfer or add photolabile protecting groups to functionalgroups, e.g., NH₂, OH, SH or the like, on a solid support. For thesemethods, conventional peptide or oligonucleotide monomers or buildingblocks having chemically removable protecting groups are used instead ofmonomers having photoprotected functional groups. In each cycle of thesynthesis procedure, the monomer is coupled to reactive sites on thesubstrate, e.g., sites deprotected in a prior photolysis step. Theprotecting group is then removed using conventional chemical techniquesand replaced with a photolabile protecting group prior to the nextphotolysis step.

A number of reagents will effect this replacement reaction. Generally,these reagents will have the following generic structure:

where R₁ is a photocleavable protecting group and X is a leaving group,i.e., from the parent acid HX. The stronger acids typically correspondto better leaving groups and thus, more reactive acylating agents.

Examples of suitable reagents are described in U.S. Patent Appl. Pub.No. 20040105932, incorporated herein by reference in its entirety forall purposes.

Conditions for carrying out this transfer are similar to those used forcoupling reaction in solid phase peptide synthesis, or for the cappingreaction in solid phase oligonucleotide synthesis. The solid phaseamine, hydroxyl or thiol groups are exposed to a solution of theprotecting group coupled to the leaving group, e.g., MeNPOC-X in anon-nucleophilic organic solvent, e.g., DMF, NMP, DCM, THF, ACN, and thelike, in the presence of a base catalysts, such as pyridine,2,6-lutidine, TEA, DIEA and the like. In cases where acylation ofsurface groups is less efficient under these conditions, nucleophiliccatalysts such as DMAP, NMI, HOBT, HOAT and the like, may also beincluded to accelerate the reaction through the in situ generation ofmore reactive acylating agents. This would typically be the case where aderivative is preferred for its longer term stability in solution, butis not sufficiently reactive without the addition of one or more of thecatalysts mentioned above. On automated synthesizers, it is generallypreferable to choose a reagent which can be stored for longer terms as astable solution and then activated with the catalysts only when needed,i.e., in the reactor system flow cell, or just prior to the addition ofthe reagent to the flow cell.

In addition to the protection of amine groups and hydroxyl groups inpeptide and oligonucleotide synthesis, the reagents and methodsdescribed herein may be used to transfer photolabile protecting groupsdirectly to any nucleophilic group, either tethered to a solid supportor in solution.

E. Individual Processing Flow Cell/Reactor System

In one embodiment, the substrate preparation process of the presentinvention is performed in a single unit operation. In this embodiment,the substrate wafer is mounted in a flow cell during, for example, boththe photolysis and chemistry or monomer addition steps. In particular,the substrate is mounted in a reactor system that allows for thephotolytic exposure of the synthesis surface of the substrate toactivate the functional groups thereon. Solutions containing chemicalmonomers are then introduced into the reactor system and contacted withthe synthesis surface, where the monomers can bind with the activefunctional groups on the substrate surface. The monomer containingsolution is then removed from the reactor system, and another photolysisstep is performed, exposing and activating different selected regions ofthe substrate surface. This process is repeated until the desiredpolymer arrays are created.

Reactor systems and flow cells that are particularly suited for thecombined photolysis/chemistry process include those described in, e.g.,U.S. Pat. No. 5,424,186 and U.S. Patent Appl. Pub. 20040105932, each ofwhich is incorporated herein by reference in its entirety for allpurposes.

Photolysis

As described above, photolithographic methods can be used to activateselected regions on the surface of the substrate. Specifically,functional groups on the surface of the substrate or present on growingpolymers on the surface of the substrate, are protected with photolabileprotecting groups. Activation of selected regions of the substrate iscarried out by exposing selected regions of the substrate surface toactivation radiation, e.g., light within the effective wavelength range,as described previously. Selective exposure is typically carried out byshining a light source through a photolithographic mask. Alternatemethods of exposing selected regions may also be used, e.g., fiber opticfaceplates and the like.

Because the individual feature sizes on the surface of the substrateprepared according to the processes described herein can typically rangeas low as 1-10 the effects of reflected or refracted light at thesurface of the substrate can have significant effects upon the abilityto expose and activate features of this size. One method of reducing theoccurrence of reflected light is to incorporate a light absorptivematerial as the back surface of the flow cell, as described above.Refraction of the light as it enters the flow cell can also result in aloss in feature resolution at the synthesis surface of the substrateresulting from refraction and reflection. To alleviate this problem,during the photolysis step, it is generally desirable to fill the flowcell with an index matching fluid (“IMF”) to match the refractive indexof the substrate, thereby reducing refraction of the incident light andthe associated losses in feature resolution. The index matching fluidwill typically have a refractive index that is close to that of thesubstrate. Typically, the refractive index of the IMF will be withinabout 10% that of the substrate, and preferably within about 5% of therefractive index of the substrate. Refraction of the light entering theflow cell, as it contacts the interface between the substrate and theIMF is thereby reduced. Where synthesis is being carried out on, e.g., asilica substrate, a particularly preferred IMF is dioxane which has arefractive index roughly equivalent to the silica substrate.

The light source used for photolysis is selected to provide a wavelengthof light that is photolytic to the particular protecting groups used,but which will not damage the forming polymer sequences. Typically, alight source which produces light in the UV range of the spectrum willbe used. For example, in oligonucleotide synthesis, the light sourcetypically provides light having a wavelength above 340 nm to effectphotolysis of the photolabile protecting groups without damaging theforming oligonucleotides. This light source is generally provided by aHg-Arc lamp employing a 340 nm cut-off filter (i.e., passing lighthaving a wavelength greater than 340-350 nm). Typical photolysisexposures are carried out at from about 6 to about 10 times the exposedhalf-life of the protecting group used, with from 8-10 times thehalf-life being preferred. For example, MeNPOC, a preferred photolabileprotecting group, has an exposed half-life of approximately 6 seconds,which translates to an exposure time of approximately 36 to 60 seconds.

Photolithographic masks used during the photolysis step typicallyinclude transparent regions and opaque regions, for exposing onlyselected portions of the substrate during a given photolysis step.Typically, the masks are fabricated from glass that has been coated witha light-reflective or absorptive material, e.g., a chrome layer. Thelight-reflective or absorptive layer is etched to provide thetransparent regions of the mask. These transparent regions correspond tothe regions to be exposed on the surface of the substrate when light isshown through the mask.

In general, it is desirable to produce arrays with smaller featuresizes, allowing the incorporation of larger amounts of information in asmaller substrate area, allowing interrogation of larger samples, moredefinitive results from an interrogation and greater possibility ofminiaturization. Alternatively, by reducing feature size, one can obtaina larger number of arrays, each having a given number of features, froma single substrate wafer. The result is substantially higher productyields for a given process. This technique, generally referred to as“die shrinking” is commonly used in the semiconductor industry toenhance product outputs or to reduce chip sizes following a over-sizedtest run of a manufacturing process.

In seeking to reduce feature size, it is important to maximize thecontrast between the regions of the substrate exposed to light during agiven photolysis step, and those regions which remain dark or are notexposed. By “contrast” is meant the sharpness of the line separating anexposed region and an unexposed region. For example, the gradient ofactivated to non-activated groups running from an activated or exposedregion to a non-exposed region is a measure of the contrast. Where thegradient is steep, the contrast is high, while a gradual gradientindicates low or poor contrast. One cause of reduced contrast is“bleed-over” from exposed regions to non-exposed regions during aparticular photolysis step. In certain embodiments, contrast betweenfeatures is enhanced through the front side exposure of the substrate.Front side exposure reduces effects of diffraction or divergence byallowing the mask to be placed closer to the synthesis surface.Additionally, and perhaps more importantly, refractive effects from thelight passing through the substrate surface prior to exposure of thesynthesis surface are also reduced or eliminated by front side exposure.

Contrast between features may also be enhanced using a number of othermethods. For example, the level of contrast degradation between tworegions generally increases as a function of the number of differentialexposures or photolysis steps between the two regions, i.e., incidenceswhere one region is exposed while the other is not. The greater thenumber of these incidences, the greater the opportunity for bleed-overfrom one region to the other during each step and the lower the level ofcontrast between the two regions. Translated into sequence information,it follows that greater numbers of differences between polymerssynthesized in adjacent regions on a substrate can result in reducedcontrast between the regions. Namely, the greater the number ofdifferences in two polymer sequences, the greater the number ofincidences of a region bearing the first polymer being exposed while theother was not. These effects are termed “edge” effects as they generallyoccur at the outer edges of the feature.

It is thus desirable to minimize these edge effects to enhance contrastin synthesis. Accordingly, in one aspect, the present invention providesa method of enhancing contrast by reducing the number of differentialsynthesis/photolysis steps between adjacent polymer sequence containingregions throughout an array.

One method of edge minimization is to divide the polymers to besequenced into blocks of related polymers, leaving blank lanes betweenthe blocks to prevent bleed-over into other blocks. While this method iseffective in reducing edge effects, it requires the creation of aspecific algorithm for each new tiling strategy. That is, the layout ofeach block in terms of probe location will depend upon the tiledsequence. In one aspect, the present invention provides methods foraligning polymer synthesis steps on an array whereby the number ofdifferential synthesis steps is reduced, and/or the syntheses inadjacent regions of the array are optimized for similarity. An exampleof a typical photolysis synthesis strategy is set forth in U.S. PatentAppl. Pub. No. 20040105932, incorporated herein by reference in itsentirety for all purposes.

Photo Acid Generator and Acid Scavenger

One embodiment of the present invention includes a photochemicalamplification method wherein photon radiation signals are converted intochemical signals in a manner that increases the effective quantum yieldof the photon in the desired reaction. The use of photochemicalamplification in methods of synthesizing patterned arrays (PASPA) isparticularly advantageous since the time and the intensity ofirradiation required to remove protective groups is decreased relativeto known direct photochemical methods. Additionally, photoacidgenerators (PAG) generate acid directly upon radiation to removeprotecting groups.

In general, radiation signals are detected by a catalyst systemincluding, for example, a photo activated catalyst (PAC). The catalystactivates an enhancer, which increases the effective quantum yield ofthe photons in subsequent chemical reactions. Such subsequent reactionsinclude the removal of protective groups in the synthesis of patternedarrays. It is desirable to remove all the protecting groups in a veryprecise location without removing protecting groups outside of thedesired location. To prevent removal of protective groups in undesirablelocations, a catalyst scavenger in some cases may be added but is notnecessary to compete for the catalyst, thus enabling the user to morespecifically define the area effected by the radiation signals.

In certain embodiments, a photo activated acid catalyst (PAAC) isirradiated. The resulting acid produced from the PAAC activates anenhancer to undergo an acid-catalyzed reaction to itself produce an acidthat removes acid labile protecting groups from a linker molecule orsynthesis intermediate. The combination of PACs and enhancers convertsand amplifies the photon signal irradiated on the surface of thesubstrate. Because of the amplification, the effective quantum yield ofthe radiation directed at the surface of the substrate is much largerthan one, resulting in high sensitivity.

One way of controlling acid catalyst “bleed-over” is the addition of anacid scavenger which serves to soak up the acid catalyst in competitionwith the photo activation reaction. Adjusting the concentration of acidcatalyst aids in fine tuning the area in which the protecting groups areremoved.

According to one embodiment of the present invention, linker moleculeshaving reactive functional groups protected by protecting groups areprovided on the surface of a substrate. A catalyst system including aPAC and an enhancer are also provided on the surface. In someembodiments, an acid catalyst scavenger may also be added. A set ofselected regions on the surface of the substrate is exposed to radiationusing well-known lithographic methods as discussed herein (See Thompsonet al. (1994) American Chemical Society, 1994:212, incorporated hereinby reference in its entirety for all purposes).

The PAC catalyst activated by the region-selective irradiation discussedabove acts to initiate a reaction of the enhancer. The enhancer producesat least one product that removes the protecting groups from the linkermolecules in the first selected regions. Preferably, the enhancer iscapble of removing protective groups in a catalytic manner. In somecases an acid scavenger may be added to react with the acid catalyst,limiting the amount of acid catalyst available to react with theenhancer. The substrate is then washed or otherwise contacted with afirst monomer that reacts with exposed functional groups on the linkermolecules. Those bound monomers are termed first-bound monomers.

A second set of selected regions is, thereafter, exposed to radiation.The radiation-initiated reactions remove the protecting groups onmolecules in the second set of selected regions, i.e., the linkermolecules and the first-bound monomers. The substrate is then contactedwith a second monomer containing a removable protective group forreaction with exposed functional groups. This process is repeated toselectively apply monomers until polymers of a desired length anddesired chemical sequence are obtained. Protective groups are thenoptionally removed and the sequence is, thereafter, optionally capped.Side chain protective groups, if present, are also optionally removed.

In one preferred embodiment, the mononomer is a 2′-deoxynucleosidephosphoramidite containing an acid removable protecting group at its 5′hydroxyl group. In an alternate embodiment, the protecting group ispresent at the 3′ hydroxyl group if synthesis of the polynucleotide isfrom the 5′ to 3′ direction. The nucleoside phosphoramidite isrepresented by the following formula:

wherein the base is adenine, guanine, thymine, cytosine or any othernucleobase analog; R₁ is a protecting group which makes the 5′ hydroxylgroup unavailable for reaction and includes dimethoxytrityl, MeNPOC,tert-butyloxycarbonyl or any of the protecting groups previouslyidentified; R₂ is cyanoethyl, methyl, t-butyl, trimethylsilyl and thelike; R₃ and R4 are isopropyl, cyclohexone and the like; and R₅ ishydrogen, NR′R″, OR, SR, CRR′R″, or OSi(R′″)₃ wherein R, R′, R″, and R′″are hydrogen, alkyl and the like. Exocyclic amines present on the basescan also be protected with acyl protecting groups such as benzoyl,isobutyryl, phenoxyacetyl and the like. The linker molecule contains anacid- or base-removable protecting group. Useful linker molecules arewell known to those skilled in the art and representative examplesinclude oligo ethers such as hexaethylene glycol, oligomers ofnucleotides, esters, carbonates, amides and the like. Useful protectinggroups include those previously listed and others known to those skilledin the art.

In another preferred embodiment, the monomer is an amino acid containingan acid- or base-removable protecting group at its amino or carboxyterminus and the linker molecule terminates in an amino or carboxy acidgroup bearing an acid- or base removable protecting group. Protectinggroups include tert-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, andany of the protective groups previously mentioned and others known tothose skilled in the art.

In a preferred embodiment the catalyst scavenger may be an acidscavenger such as an amine and more specifically may be trioctylamine or2,5-di-tertbutylanaline. Other acid scavengers include carboxylate saltsand hydroxides. See, e.g. Huang (1999) Proc. SPIE-Int. Soc. Opt. Eng.3678:1040, incorporated herein by reference in its entirety for allpurposes. Those of skill in the art will be familiar with other acidscavengers which will be appropriate for the present invention.

In another preferred embodiment, the catalyst scavenger may be a basescavenger such as acetic acid or trichloro acetic acid. Other basescavengers include phosphoric acid, sulfuric acid or any othercarboxylic acid. Care should be taken to chose a base scavenger whichwill not interfere with or destroy the monomer. Those of skill in theart will be familiar with other base scavengers which will beappropriate for the present invention.

It is apparent to those skilled in the art that photochemicallyamplified radiation-based activation is not limited to photo activatedenhancers or catalysts or to acid or base production cascades. Variouscompounds or groups can produce catalysts or enhancers in response toradiation exposure. Non-limiting examples include photogeneration ofradicals using diphenylsulfide, benzoylperoxide,2,2′-azobis(butyronitrile), benzoin and the like; cations such astriarylsulfonium salts, diaryl iodonium salts and the like; and anions.Furthermore, it is apparent to those of skill in the art that thecatalyst scavengers are not limited to acid or base scavengers but mayinclude any other compound which will interfere with the catalysts'ability to interact with the enhancer.

In a preferred embodiment, the catalyst and catalyst scavenger arecapable of engaging in a cyclic reaction. For example, a compound Xcomprises subcompound Y which is capable of acting as a catalyst andsubcompound Z which is capable of acting as a catalyst scavenger.Compound X is capable of entering into an excited state after exposureto radiation. During this excited state the subcompounds Y and Zseparate and subcompound Y is free to catalyze removal of protectinggroups. In a further preferred embodiment, the subcompounds Y and Z arecapable of remaining in this excited state for only a very short periodof time. This time period may be from between a few nanoseconds to a fewmilliseconds. After the time period lapses, subcompounds Y and Z arefree to interact with one another once again forming compound X Exposureto radiation may then initiate another cycle. In a preferred embodiment,compound X is very stable prior to exposure to radiation, and onlycapable of interacting with other molecules during the excited state.

The selection of radiation sources is based upon the sensitivityspectrum of the compound to be irradiated. Potential damage to synthesissubstrates, intermediates, or products is also considered. In somepreferred embodiments, the radiation could be ultraviolet (UV), infrared(IR), or visible light. In a specific embodiment, the radiation sourceis a light beam with a wavelength in the range of from 190-500 nm,preferably from 250-450 nm, more preferably from 365-400 nm. Specificradiation wavelengths include 193 nm, 254 nm, 313 nm, 340 nm, 365 nm,396 nm, 413 nm, 436 nm, and 500 nm. Suitable light sources include highpressure mercury arc lamps and are readily commercially available fromOriel, OAI, Cannon, A-B Manufacturing and the like. In embodimentsutilizing the catalytic system, the sensitivity spectrum of the RAC canbe altered by providing radiation sensitizers. The energy of thesensitizer must be matched to the PAC and include2-ethyl-9,10-dimethoxy-anthracene, perylene, phenothiazine, xanthone andthe like. Many radiation sensitizers are known to those skilled in theart and include those previously mentioned. It is to be understood thatone of ordinary skill in the art will be able to readily identifyadditional radiation sensitizers based upon the present disclosure.

In addition to the foregoing, aspects of the invention includeadditional methods which can be used to generate an array ofoligonucleotides on a single substrate are described in U.S. Pat. Nos.5,677,195 and 5,384,261, and in PCT Publication No. WO 93/09668, each ofwhich is incorporated herein by reference in its entirety for allpurposes. In the methods disclosed in these applications, reagents aredelivered to the substrate by either (1) flowing within a channeldefined on predefined regions or (2) “spotting” on predefined regions or(3) through the use of photoresist. However, other approaches, as wellas combinations of spotting and flowing, may be employed. In eachinstance, certain activated regions of the substrate are mechanicallyseparated from other regions when the monomer solutions are delivered tothe various reaction sites.

In one aspect, a typical “flow channel” method is applied to thecompounds and libraries of the present invention, and can generally bedescribed as follows. Diverse polymer sequences are synthesized atselected regions of a substrate or solid support by forming flowchannels on a surface of the substrate through which appropriatereagents flow or in which appropriate reagents are placed. For example,assume a monomer “A” is to be bound to the substrate in a first group ofselected regions. If necessary, all or part of the surface of thesubstrate in all or a part of the selected regions is activated forbinding by, for example, flowing appropriate reagents through all orsome of the channels, or by washing the entire substrate withappropriate reagents. After placement of a channel block on the surfaceof the substrate, a reagent having the monomer A flows through or isplaced in all or some of the channel(s). The channels provide fluidcontact to the first selected regions, thereby binding the monomer A onthe substrate directly or indirectly (via a spacer) in the firstselected regions.

Thereafter, a monomer B is coupled to second selected regions, some ofwhich may be included among the first selected regions. The secondselected regions will be in fluid contact with a second flow channel(s)through translation, rotation, or replacement of the channel block onthe surface of the substrate; through opening or closing a selectedvalve; or through deposition of a layer of chemical or photoresist. Ifnecessary, a step is performed for activating at least the secondregions. Thereafter, the monomer B is flowed through or placed in thesecond flow channel(s), binding monomer B at the second selectedlocations. In this particular example, the resulting sequences bound tothe substrate at this stage of processing will be, for example, A, B,and AB. The process is repeated to form a vast array of sequences ofdesired length at known locations on the substrate.

After the substrate is activated, monomer A can be flowed through someof the channels, monomer B can be flowed through other channels, amonomer C can be flowed through still other channels, etc. In thismanner, many or all of the reaction regions are reacted with a monomerbefore the channel block must be moved or the substrate must be washedand/or reactivated. By making use of many or all of the availablereaction regions simultaneously, the number of washing and activationsteps can be minimized.

One of skill in the art will recognize that there are alternativemethods of forming channels or otherwise protecting a portion of thesurface of the substrate. For example, according to some embodiments, aprotective coating such as a hydrophilic or hydrophobic coating(depending upon the nature of the solvent) is utilized over portions ofthe substrate to be protected, sometimes in combination with materialsthat facilitate wetting by the reactant solution in other regions. Inthis manner, the flowing solutions are further prevented from passingoutside of their designated flow paths.

In another aspect, the “spotting” methods of preparing compounds andlibraries of the present invention can be implemented in much the samemanner as the flow channel methods. For example, a monomer A can bedelivered to and coupled with a first group of reaction regions whichhave been appropriately activated. Thereafter, a monomer B can bedelivered to and reacted with a second group of activated reactionregions. Unlike the flow channel embodiments described above, reactantsare delivered by directly depositing (rather than flowing) relativelysmall quantities of them in selected regions. In some steps, of course,the entire substrate surface can be sprayed or otherwise coated with asolution. In preferred embodiments, a dispenser moves from region toregion, depositing only as much monomer as necessary at each stop.Typical dispensers include a micropipette to deliver the monomersolution to the substrate and a robotic system to control the positionof the micropipette with respect to the substrate. In other embodiments,the dispenser includes a series of tubes, a manifold, an array ofpipettes, or the like so that various reagents can be delivered to thereaction regions simultaneously.

F. Assembly of Probe Array Cartridges

Following synthesis, final dcprotection and other finishing steps, e.g.polymer coat removal where necessary, the substrate wafer can beassembled for use as individual substrate segments. Assembly typicallyemploys the steps of separating the substrate wafer into individualsubstrate segments, and inserting or attaching these individual segmentsto a housing which includes a reaction chamber in fluid communicationwith the front surface of the substrate segment, e.g., the surfacehaving the polymers synthesized thereon.

Methods of separating and packaging substrate wafers are described insubstantial detail in Published PCT Application No. 95/33846, which ishereby incorporated herein by reference in its entirety for allpurposes.

Typically, the arrays are synthesized on the substrate wafer in a gridpattern, with each array being separated from each other array by ablank region where no compounds have been synthesized. These separatingregions are termed “streets.” The wafer typically includes a number ofalignment marks located in these streets. These marks serve a number ofpurposes, including aligning the masks during synthesis of the arrays asdescribed above, separation of the wafer into individual chips andplacement of each chip into its respective housing for subsequent use,which are both described in greater detail below.

Generally, the substrate wafer can be separated into a number ofindividual substrates using scribe and break methods that are well knownin the semiconductor manufacturing industry. For example, well knownscribe and break devices may be used for carrying out the separationsteps, e.g., a fully programmable computer controlled scribe and breakdevices, such as a DX-III Scriber-Breaker manufactured by DynatexInternational (Santa Rosa, Calif.), or the LCD-1 scriber/dicermanufactured by Loomis Industries Inc. (St. Helena, Calif.). The stepstypically involve scribing along the desired separation points, e.g.,between the individual synthesized arrays on the substrate wafersurface, followed by application of a breaking force along the scribeline. For example, typical scribe and break devices break the wafer bystriking the bottom surface of the wafer along the scribe lines with animpulse bar, or utilizing a three point beam substrate bendingoperation. The shock from the impulse bar fractures the wafer along thescribe line. Because the majority of force applied by the impulse bar isdissipated along the scribe line, the device is able to provide highbreaking forces without exerting significant force on the substrateitself, allowing separation of the wafer without damaging the individualchips.

In alternative methods, the wafer may be separated into individualsegments by, e.g., sawing methods, such as those described in U.S. Pat.No. 4,016,855, incorporated herein by reference in its entirety for allpurposes.

Once the wafer is separated into individual segments, these segments maybe assembled in a housing that is suited for the particular analysis forwhich the array will be used. Examples of methods and devices forassembling the substrate segments or arrays in cartridges are describedin, e.g., U.S. Pat. No. 5,945,334, incorporated herein by reference inits entirety for all purposes. Typically, the housing includes a bodyhaving a cavity disposed within it. The substrate segment is mountedover the cavity on the body such that the front side of the segment,e.g., the side upon which the polymers have been synthesized, is influid communication with the cavity. The bottom of the cavity mayoptionally include a light absorptive material, such as a glass filteror carbon dye, to prevent impinging light from being scattered orreflected during imaging by detection systems. This feature improves thesignal-to-noise ratio of such systems by significantly reducing thepotential imaging of undesired reflected light.

The cartridge also typically includes fluid inlets and fluid outlets forflowing fluids into and through the cavity. A septum, plug, or otherseal may be employed across the inlets and/or outlets to seal the fluidsin the cavity. The cartridge also typically includes alignmentstructures, e.g., alignment pins, bores, and/or an asymmetrical shape toensure correct insertion and/or alignment of the cartridge in theassembly devices, hybridization stations, and reader devices. Example ofcertain embodiments of cartridges are described in U.S. Patent Appl.Pub. No. 20040105932, incorporated herein by reference in its entiretyfor all purposes.

In a preferred embodiment, the bottom casing with selected cavitydimensions may be removed from the middle and top casings, and replacedwith another bottom casing with different cavity dimensions. This allowsa user to attach a chip having a different size or shape by changing thebottom casing, thereby providing ease in using different chip sizes,shapes, and the like. Of course, the size, shape, and orientation of thecavity will depend upon the particular application. The body of thecartridge may generally be fabricated from one or more parts made usinga number of manufacturing techniques. In preferred aspects, thecartridge is fabricated from two or more injection molded plastic parts.Injection molding enables the casings to be formed inexpensively. Also,assembling the cartridge from two parts simplifies the construction ofvarious features, such as the internal channels for introducing fluidsinto the cavity. As a result, the cartridges may be manufactured at arelatively low cost.

The substrate segment may be attached to the body of the cartridge usinga variety of methods. In preferred aspects, the substrate is attachedusing an adhesive. Preferred adhesives are resistant to degradationunder conditions to which the cartridge will be subjected. Inparticularly preferred aspects, an ultraviolet cured adhesive attachesthe substrate segment to the cartridge. Devices and methods forattaching the substrate segment are described in Published PCTApplication No. 95/33846, incorporated herein by reference in itsentirety for all purposes. Particularly preferred adhesives arecommercially available from a variety of commercial sources, includingLoctite Corp. (Irvine, Calif.) and Dymax Corp. (Torrington, Conn.).

A variety of modifications can be incorporated in the assembly methodsand devices that are generally described herein, and these too areoutlined in greater detail in published PCT Application No. 95/33846,incorporated herein by reference in its entirety for all purposes.

Upon completion, the cartridged substrate will have a variety of uses.For example, the cartridge can be used in a variety of sequencing byhybridization (“SBH”) methods, sequence checking methods, diagnosticmethods and the like. Arrays which are particularly suited for sequencechecking and SBH methods are described in, e.g., U.S. patent applicationSer. Nos. 08/505,919, filed Jul. 24, 1995, 08/441,887, filed May 161995, 07/972,007, filed Nov. 5, 1992, each of which is incorporatedherein by reference in its entirety for all purposes.

Typically, in carrying out these methods, the cartridged substrate ismounted on a hybridization station where it is connected to a fluiddelivery system. The fluid delivery system is connected to the cartridgeby inserting needles into the inlet and outlet ports through the septadisposed therein. In this manner, various fluids are introduced into thecavity for contacting the probes synthesized on the front side of thesubstrate segment, during the hybridization process.

Usually, hybridization is performed by first exposing the sample with apre-hybridization solution. Next, the sample is incubated under bindingconditions for a suitable binding period with a sample solution that isto be analyzed. The sample solution generally contains a targetmolecule, e.g., a target nucleic acid, the presence or sequence of whichis of interest to the investigator. Binding conditions will varydepending on the application and are selected in accordance with thegeneral binding methods known including those referred to in: Maniatiset al Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., ColdSpring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc.,San Diego, Calif.; Laboratory Techniques in Biochemistry and MolecularBiology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen,ed. Elsevier, N.Y., (1993); and Young and Davis (1983) Proc. Natl. Acad.Sci. USA 80:1194, each of which is incorporated herein by reference inits entirety for all purposes. In certain embodiments, the solution maycontain about 1 M salt and about 1 to 50 nM targets. Optionally, thefluid delivery system includes an agitator to improve mixing in thecavity, which shortens the incubation period. Finally, the sample iswashed with a buffer, which may be 6× SSPE buffer, to remove the unboundtargets. In some embodiments, the cavity is filled with the buffer afterwashing the sample.

Following hybridization and appropriate rinsing/washing, the cartridgedsubstrate may be aligned on a detection or imaging system, such as thosedisclosed in U.S. Pat. Nos. 5,143,854 and 5,631,734, and U.S. patentapplication Ser. Nos. 08/465,782, filed Jun. 6, 1995, and 08/456,598,filed Jun. 1, 1995, each of which is incorporated herein by reference inits entirety for all purposes. Such detection systems may take advantageof the cartridge's asymmetry (i.e., non-flush edge) by employing aholder to match the shape of the cartridge specifically. Thus, thecartridge is assured of being properly oriented and aligned forscanning. The imaging systems are capable of qualitatively analyzing thereaction between the probes and targets. Based on this analysis,sequence information of the targets is extracted. In accordance with apreferred embodiment of the present invention, confocal fluorescencescanning is conducted front side, since the excitation light wouldotherwise be blocked by the substrate.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. All publications and patent documents cited in thisapplication are incorporated by reference in their entirety for allpurposes to the same extent as if each individual publication or patentdocument were so individually denoted.

1. A method of performing confocal laser microscopy on a polymer arraydisposed on a silicon wafer substrate, said method comprising the stepsof: providing a silicon wafer substrate having a top side and a bottomside; coating said top side of said silicon wafer with an oxide coatingto provide an oxide coated wafer; covalently coupling a plurality ofprobes to said top side of said coated wafer to provide a fixed polymerarray; hybridizing said fixed polymer array with a plurality of labeledligands; and assaying for one or more hybridized ligands using confocallaser fluorescence microscopy to detect hybridization.
 2. The method ofclaim 1, further comprising applying BisB to said oxide coating.
 3. Amethod of performing confocal laser microscopy on a polymer arraydisposed on a silicon wafer substrate, said method comprising the stepsof: providing a silicon wafer substrate having a top side and a bottomside; coating said top side of said substrate with a transparent oxidelayer to provide an oxide coated wafer; depositing a reactive functionalgroup comprising a labile protecting group substantially uniformlyacross the transparent oxide layer; selectively removing one or more ofsaid labile protecting groups from predefined regions of said wafer toprovide exposed functional groups in said predefined regions; reactingsaid exposed functional groups with a monomer comprising a reactivefunctional group and a labile protecting group; repeating the steps ofselectively removing and reacting to produce said polymer array;hybridizing said polymer array with a plurality of ligands; and assayingfor one or more hybridized ligands using a confocal laser fluorescencemicroscopy to detect hybridization.
 4. The method of claim 3, whereinsaid oxide layer has a thickness of at least 3,500 angstroms.
 5. Themethod of claim 3, wherein said oxide layer has a thickness of at least35,000 angstroms.
 6. The method of claim 3, wherein said labileprotecting group is an acid labile protecting group.
 7. The method ofclaim 6, wherein said acid labile protecting group is a dimethoxytritylgroup.
 8. The method of claim 6, wherein said acid labile protectinggroup is removed by activating a photoacid generator with light of anappropriate wavelength to produce acid.
 9. The method of claim 8,wherein said photoacid generator is an ionic photoacid generator or anon-ionic photoacid generator.
 10. The method of claim 9, wherein saidphotoacid generator is an ionic photoacid generator.
 11. The method ofclaim 9, wherein said photoacid generator is a non-ionic photoacidgenerator.
 12. The method of claim 11, wherein said non-ionic photoacidgenerator is 2,6-dinitrobenzyl tosylate.
 13. The method of claim 10,wherein said ionic photoacid generator is an onium salt.
 14. The methodof claim 13, wherein said onium salt is bis-(4-t-butyl phenyl) iodoniumPF₆ ⁻.
 15. The method of claim 3, wherein said labile protective groupis a photolabile protecting group.
 16. The method of claim 3, whereinsaid monomer is selected from the group consisting of a nucleotide, anucleic acid, an amino acid and a peptide.
 17. The method of claim 3,wherein said monomer is a nucleic acid and said labile protecting groupis MeNPOC.
 18. The method of claim 3, wherein said monomer is a nucleicacid and said labile protecting group is NNPOC or MBPMOC.